Samples and sequencing. DNA from Isle Royale wolves was extracted from blood samples archived at Michigan Technological University. Before this study, the Isle Royale population was not actively managed, and human interventions (e.g., trapping and fitting radio collars) were kept to a minimum. Thus, many wolf samples have been collected opportunistically over the years from urine, scat, and carcasses, and complete lifetime survival and reproduction data are generally not available. Of the DNA samples present in the archives, those with the highest concentration and quality were selected for sequencing to ensure high-quality sequence data and high coverage. DNA from Minnesota and Canadian Arctic wolves was extracted from blood and tissue samples from the archive of R.K.W. that were used in previous studies (34, 58). Although our sample of Minnesota wolves may not be directly derived from the ancestral population for Isle Royale wolves, a previous analysis of 3 Isle Royale and 11 Minnesota wolves at >44,000 SNP loci suggests that the two populations have very high genetic similarity (34). Whole-genome sequencing was performed on an Illumina HiSeq 4000 at the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley. Previously sequenced genomes with high coverage were downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (see table S1).

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